Human serum

To profile the SARS-CoV-2 specific T effector subsets in COVID-19 patients, frozen PBMCs from the first convalescent timepoint (median 19.5 days PIO, IQR 16–26) were thawed and rested overnight at 37 °C in RPMI 1640 (Hyclone) supplemented with 5% human serum (Innovative Research), followed by stimulation with phorbol 12-myristate 13-acetate (PMA 100 ng/mL, Sigma Aldrich) and ionomycin (1 µg/mL, Sigma Aldrich), or pooled SARS-CoV-2 PepTivator S, S1, M and N peptides (0.6 nmol/mL each) (Miltenyi Biotec) for 6 h. Brefeldin A and Monesin (1 × , ThermoFisher Scientific) were added at 2 h post-stimulation. Cells were stained for surface markers in the dark at room temperature for 30 min (Supplemental Table 4), followed by fixation and permeabilization for 30 min with Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher Scientific). Permeabilized cells were then stained for intracellular cytokines in the dark at room temperature for 30 min (Supplemental Table 4). Cells were then washed with PBS and centrifuged at 800 × g for 5 min before transferring to respective polystyrene FACS tubes containing 5 μL (5.4 × 10³ beads) of CountBright Absolute Counting Beads (Invitrogen). Cells were acquired with the Cytek Aurora cytometer (Cytek Biosciences) and analyzed using FlowJo (Tree Star).

The cell-ELISA was performed as described with modifications^{13 (link)}. Briefly, Cells were seeded at 10,000 cells/well in 96-well culture plates and incubated overnight at 37 °C in 5% CO₂. Cells were then cultured in media containing 10% pooled human serum (Innovative Research, Novi, MI) for 1 h to activate the complement system. Plates were washed with phosphate-buffered saline (PBS) followed by fixing with 3% paraformaldehyde (PFA) for 15 min. The cells were incubated in blocking buffer (5% skim milk in Tris-buffered saline; TBS) for 1 h at 37 °C. A rabbit polyclonal C5b-9 antibody (Abcam, Cambridge, MA) diluted in blocking buffer (1:4,000) was added to the plate, and incubated with the cells for 2 h. The plate was washed three times with TBS/T for 15 min, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Health Care, Buckinghamshire, UK) was added. After incubation at room temperature for 1 h, the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, KPL, Gaithersburg, MD) was used. The absorbance at 450 nm was measured by a microplate reader (Molecular Devices, Silicon Valley, CA).

Monocytes were cultured in Iscove's Modified Dulbecco's Medium (IMDM; Hyclone) supplemented with 5% human serum (Innovative Research) and 1% penicillin/streptomycin (Invitrogen) in a humidified 37°C incubator with 5% CO₂. For lipopolysaccharide (LPS) stimulation, 100 ng/ml LPS (E. coli serotype O111:B4) was added to the culture medium for the indicated duration.

The PG was extracted and analyzed according to B. Glauner (1988). Briefly, A. baumannii strains were grown in LB or 50% heat‐inactivated human serum (Innovative Research) to ~10⁸ CFU ml^–1. Cells were then collected by centrifugation, resuspended in 6 ml ice‐cold water and lysed by drop wise addition to 6 ml boiling 8% SDS. The PG was purified and digested with the muramidase cellosyl (Hoechst, Frankfurtam Main, Germany) to release the muropeptides, which were reduced by sodium borohydride, and separated on a Prontosil 120‐3‐6C18 AQ reversed phase column (Bischoff, Leonberg, Germany). The eluted muropeptides were detected by their absorbance at 205 nm.